Enhanced time-saving extraction procedure for the analysis

of fecal fat by Fourier transform infrared spectroscopy.

To the Editor:

Recently, we presented an improved procedure for the determination of fecal fat by means of
Fourier transform infrared spectroscopy (1). This method can be used in laboratories equipped with
a mid-infrared spectrometer. With this method, fecal fat was extracted from stool samples with
petroleum ether-ethanol. After extraction, the petroleum ether was dried, and the fatty acids were
redis-solved in chloroform before measurement. Because the extraction procedure in the
previously described analytical method (1) was still rather time-consuming, we replaced the
petroleum ether-ethanol extraction with a single chloroform extraction.
In the new extraction procedure, 1 g of homogenized stool sample was suspended in 2.5 mL of
water. After the suspension was mixed, 0.5 mL of 12 mol/L HCl and 2.0 mL of chloroform (gradient
grade) were added, and the sample was shaken vigorously for 10 min. At this stage, the samples
were either stored at -20[degrees]C or analyzed immediately. The extract was centrifuged for 5 min
at 3000g at room crude fat analyzer temperature, after which the organic layer was transferred to a
transmission cell (pathlength, 0.05 mm) with calcium fluoride crystals.
Spectra (n = 111) were scanned in the mid-infrared region from 4000 to 650 [cm.sup.-1] with a
Perkin-Elmer Spectrum 2000 spectrometer (PerkinElmer). Calibration was performed with a
mixture of stearic and palmitic acid (65:35, by weight) ranging from 0 to 150 g/kg (1). For both the
"old" and the "new" extraction procedures, the spectroscopic band at 2855 [cm.sup.-1] (C--H
symmetric stretch vibration) was used to calculate the amount of fat (g/kg). Passing and Bablok
regression was performed for method agreement.

The results obtained by the two methods showed good agreement (r =0.991; Fig. 1). By Passing and
Bablok regression, the slope was 1.055(range, 1.026-1.088), the intercept was 0.241 (range, 0.1810.296), andthe standard deviation of the residuals ([S.sub.y|x].) was 0.365. Forthe new extraction
procedure, the intra-and interassay imprecision (asthe CVs; n = 10) was 4.0% and 5.0%,
respectively, for a stool samplecontaining 43 g/kg fat. For a sample with 26 g/kg fat, the intraandinterassay CVs were 3.9% and 10%, respectively. Recovery of thestearic-palmitic acid (65:35, by
weight) calibrator added to stool was>95% with the new extraction procedure. The majority of fecal
fat iscomposed mainly of [C.sub.16:0] and [C.sub.18:0] free fatty acids, whichcan be extracted easily
from the stool with the new extraction method.The reduction in analysis time gained in this way is
~2.5 h for a seriesof 10 stools.
We conclude that the new simplified extraction procedure for fecal fat determination gives
comparable results to the old extraction procedure and allows considerable reduction in analysis
time, use of chemicals, and technical equipment.
[FIGURE 1 OMITTED]
Reference

(1.) Jakobs BS, Volmer M, Swinkels DW, Hofs MTW, Donkervoort S, Joosting MMJ, et al. New
method for faecal fat determination by mid-infrared spectroscopy, using a transmission cell: an
improvement in standardization. Ann Clin Biochem 2000;37:343-9.

Bernadette S. Jakobs [1] *

Marcel Volmer [2]

Marc T.W. Hofs [1]
Dorine W. Swinkels [1]
[1] Department of Clinical Chemistry University Hospital Nijmegen St. Radboud PO Box 9101 6500
HB Nijmegen, The Netherlands
[2] Department of Clinical Chemistry University Hospital Groningen PO Box 30.001 9700 RB
Groningen, The Netherlands
* Author for correspondence. E-mail B. Jakobs@ckcl.azn.nl.